CYP induction, regulatory

The test compounds are incubated with plated cryopreserved human hepatocytes (3 individual donors) after which the cells are lysed, and total RNA is isolated. Target mRNA-levels (CYP1A2, CYP2B6, CYP3A4) are then analyzed by real-time quantitative PCR. Possible toxicity of the study compounds towards the cells is monitored visually during the study. The experiment includes pre-study cytotoxicity assessment (ATP+LDH) & visual solubility check and test compound stability assessment. In addition, determination of unbound fraction in incubation medium and/or human plasma and CYP activity endpoint with UPLC/MS/MS analysis can be included.
check Protocol

Test concentrations: 6

Time points: 48h

Replicates: 3

Positive controls

RT-qPCR analysis

check Deliverables

Induction (xx-fold) based on CYP-mRNA levels and optionally also based on CYP-activity levels, EC50 and Emax values if applicable, cytotoxicity and visual solubility evaluation, test compound stability, optionally free fraction of the test compound and CYP activity endpoint

check Reporting

Full Word/PDF report

Background

Cytochrome P450 (CYP) induction by a new chemical entity results in the increased amount and activity of relevant drug-metabolizing CYPs, which may lead to enhanced metabolism of co-medicated drugs. CYP induction is a relatively slow phenomenon due to gene regulation events, such as nuclear receptor activation and synthesis of mRNA and enzyme. Exposure times for the assays are usually 24 – 72 hours, followed by PXR activation, mRNA and/or CYP activity assessment. As a part of our drug-drug interactions services, the CYP induction screening assays are tailored for cost-efficient simultaneous evaluation of higher number of compounds, whereas the regulatory compliant induction assay with human hepatocytes is a comprehensive package required by FDA and EMA. Each assay delivers CYP induction relative to vehicle control (x-fold) and / or EC50 values if feasible (concentration where 50 % of maximal induction is achieved). The induction assays are available for CYP1A2, CYP2B6 and CYP3A4. Assays for other CYPs are available upon request.

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